During the lifetime of an HCV infected patient, interplay occurs between innate immunity of the host and virus evolution. The innate responses play an important role in controlling pathogenesis and disease severity. The binding of either viral replicates or protein products to specific cellular pathogen recognition receptors, such as Toll-like receptor 1 (TLR1) and RIG-I, triggers the innate antiviral response. The HCV NS3/4A serine protease disrupts innate immunity signaling pathways via proteolytic cleavage of critical adaptor molecules such as TRIF and IPS1. These pathways are essential for Type I IFN induction, and provide a mechanism by which HCV subverts innate immunity and establishes persistent infection. During our study of HCV genetic evolution during natural human infection, we have isolated novel and interesting NS3/4A mutants associated with mild and severe disease phenotype in vivo. We now propose to characterize the biochemistry and molecular biology of such naturally occurring NS3/4A variants, as they evolve in humans over time during diverse clinical outcomes. The first Aim will amplify and sequence NS3/4A genetic cassettes from, 5 mild and 5 severe disease patients, archived viremic specimens previously obtained from our 1,200 Alaskan Natives and American Indian cohort (AN/AI), which is in it's 12th year of contiguous NIH funding to describe in vivo viral- host dynamics during untreated, long term naturally occurring chronic hepatitis C. We will clone and express viral enzyme complexes of interest for functional studies, on a case-by case basis. In collaboration with our institutional colleague, Dr. Michael Gale Jr., an expert on human innate immunity and the biochemistry of HCV NS3/4A, Aim 1 will also compare protease biochemistry of natural variant NS3/4A complexes with prototype complexes, including stability, substrate recognition (including both viral and host targets) and enzyme kinetics. Aim 2 will look at protease biological function against innate immunity using the trans-rescue approach. We will thus describe natural evolution of NS3/4A genes and enzyme function in untreated humans over time, comparing activities at early and late times post infection. We hypothesize that genetic variants of NS3/4A, isolated from patients with severe disease, will display different in vivo evolutionary dynamics, will differ in viral polyprotein processing, and will exert broader and more efficient control of host innate immune pathways compared to protease variants from mild disease cases. This proposal is significant because it describes and tests the potential pathogenic significance of naturally occurring mutations in HCV genomes during mild and severe disease progression.